1. Field of the Invention
This invention relates to the regulation of plant growth, and more particularly to the molecular cloning and expression of a gibberellin 20-oxidase gene and its use, for example in transgenic plants.
2. Description of the Related Art
Chemical compounds for control of plant growth have been in commercial use for many years. Many of these compounds act by inhibiting various steps in the biosynthesis of gibberellins (GAs). GAs form a large group of diterpenoid natural products, some members of which function as hormones in plants, controlling many aspects of development, including, for example, shoot elongation. Among the groups of compounds which compounds, compounds with a nitrogen-containing heterocycle, and acylcyclohexanediones. However, the use of such chemicals involves several problems. It is, for example, difficult to apply the chemicals to plants in the appropriate quantities, or to select plant organs, without the chemicals spreading to other plants or animal life. There is a risk of persistence which can make it difficult to grow other crops subsequently to treated crops. A problem addressed by the present invention is therefore to avoid the use of such chemicals. This problem can be solved within this application by providing means for plant growth control at the plant gene level.
The later steps of the GA biosynthetic pathway are catalysed by soluble 2-oxoglutarate-dependent dioxygenases, several of which have been proposed as regulatory enzymes in the biosynthesis of the physiologically important C19 compound, GA1. For example, the activity of the GA 20-oxidase is enhanced by long days in certain photoperiod-sensitive plants and is down-regulated as a consequence of GA1 action in several species.
According to the invention, there is provided a DNA sequence which encodes a polypeptide exhibiting GA 20-oxidase activity. This disclosure is the first example of the molecular cloning of a GA:2-oxoglutarate dioxygenase. The enzyme GA 20-oxidase is also known as a 20-hydroxylase or C-20 oxidase, as it catalyses oxidation reactions at the C-20 carbon atom of the GA structure. It is a dioxygenase, as oxoglutarate is simultaneously oxidised.
As demonstrated in the Examples the DNA sequence of the present invention encodes GA 20-oxidase capable of acting essentially on one or more of the following substrates: GA12, GA53, GA15 (open or closed lactone), GA44 (open or closed lactone), GA24, GA19 and GA23 among others.
The present invention thus further relates to a DNA sequence encoding a polypeptide exhibiting GA 20-oxidase activity, in which the polypeptide exhibiting GA 20-oxidase activity is capable of acting essentially on one or more of the following substrates: GA12, GA53, GA15 (open or closed lactone), GA44 (open or closed lactone), GA24, GA19 and GA23 
The DNA sequence of the invention may encode a GA 20-oxidase from, in principle, any plants or fungi, but preferably from monocotyledonous and dicotyledonous plants, and more preferably from dicotyledonous plants. A particularly suitable source is plants of the family Cucurbitaceae, such as C. maxima, of which the immature seeds are a convenient source. A further suitable source is plants of the family Cruciferae, such as Arabidopsis thaliana, of which shoot material is a convenient source.
A preferred embodiment of the invention is therefore a DNA sequence which encodes a GA 20-oxidase obtainable from plants or fungi, preferably from monocotyledonous and dicotyledonous plants respectively, more preferably from dicotyledonous plants and most preferably from plants of the family Cucurbitaceae and Cruciferae respectively, such as C. maxima and Arabidopsis thaliana, or a protein having substantial homology thereto.
As used in the present application, substantial sequence homology means close structural relationship between sequences of nucleotides or amino acids. For example, substantially homologous DNA sequences may be 60% homologous, preferably 80% and most preferably 90% or 95% homologous, or more, and substantially homologous amino acid sequences may preferably be 35%, more preferably 50%, most preferably more than 50% homologous. Homology also includes a relationship wherein one or several subsequences of nucleotides or amino acids are missing, or subsequences with additional nucleotides or amino acids are interdispersed.
The term xe2x80x9chomologyxe2x80x9d as used herein not only embraces structural homology but also functional homology.
The invention thus further relates to a DNA sequence, which encodes a GA 20-oxidase obtainable from Cucurbita maxima or Arabidopsis thaliana or a protein having at least 35%, preferably at least 50%, and most preferably more than 50% homology therewith.
More specifically, the invention relates to a DNA having a sequence corresponding to the open reading frame of the sequence shown in SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5, or an equivalent sequence through the degeneracy of the genetic code, including derivatives capable of hybridizing with the sequence shown in SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5, and still encoding a polypeptide exhibiting GA 20-oxidase activity.
A preferred embodiment of the invention is therefore a substantially pure DNA as shown in SEQ ID NO 1, SEQ ID NO 3, or SEQ ID NO 5, or having substantial sequence homology to the sequence shown in SEQ ID NO 1, SEQ ID NO 3, or SEQ ID NO 5.
The DNA sequence according to the invention is preferably a recombinant DNA comprising a DNA sequence which encodes a recombinant polypeptide exhibiting GA 20-oxidase activity. In one embodiment of the invention the recombinant DNA is in the form of a cDNA clone.
It is a further object of the invention to provide a chimaeric gene construct comprising a DNA sequence encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with plant expression signals including promoter and termination sequences capable of causing the gene to express a polypeptide exhibiting GA 20-oxidase activity within a plant, wherein the promoter sequences are preferably those of an inducible promoter or a tissue-preferential or a tissue-specific promoter.
The invention further comprises a chimaeric gene construct comprising at least a part of a reverse GA 20-oxidase nucleotide sequence, in operable linkage with plant expression signals including promoter and termination sequences capable of causing the reverse sequence to express antisense mRNA within a plant.
It is also an object of the invention to provide transformed host cells comprising recombinant DNA encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with expression signals including promoter and termination sequences which permit expression of said DNA in the host cell.
A preferred embodiment of the invention is a transgenic plant including seed and progeny or propagules thereof comprising preferably stably integrated into its genome a chimeric gene construct as mentioned hereinbefore. Preferred is a monocotyledonous and a dicotyledonous plant, respectively such as tobacco, tomato, cotton, sunflower, maize, wheat and Dactylis glomerata. 
Especially preferred is a transgenic plant which is a monocotyledonous plant, preferably a maize plant or a wheat plant
The invention also comprises a recombinant polypeptide obtainable from plants or fungi exhibiting GA 20-oxidase activity, which polypeptide is preferably capable of acting essentially on one or more of the following substrates: GA12, GA53, GA15 (open or closed lactone), GA44 (open or closed lactone), GA24, GA19 and GA23.
A preferred embodiment of the invention is therefore a recombinant polypeptide which exhibits a GA 20-oxidase activity and which is obtainable from plants or fungi, preferably from monocotyledonous and dicotyledonous plants, respectively, more preferably from dicotyledonous plants and most preferably from plants of the family Cucurbitaceae and Cruciferae respectively, such as C. maxima and Arabidopsis thaliana, or a protein having substantial homology thereto.
More specifically, the invention relates to a recombinant polypeptide having a sequence as shown in SEQ ID NO 2, SEQ ID NO 4 AND SEQ ID NO 6, or an sequence that is substantially homolgous thereto.
The invention further comprises a method of preparing a DNA sequence encoding a GA 20-oxidase, comprising preparing a cDNA library from a suitable source organism, and screening this library by means of one of the conventionally applied screening systems.
The invention also comprises a method of preparing a recombinant polypeptide exhibiting GA 20-oxidase activity, which comprises of one of the DNA sequences mentioned hereinbefore.
A further embodiment of the invention is a method of identifying DNA sequences comprising a DNA region encoding a polypeptide exhibiting GA 20-oxidase activity which method comprises preparing a cDNA or a genomic library from a suitable source organism and screening this library by means of hybridisation using a suitable DNA as a hybridisation probe.
In the first place, the present invention relates to a DNA sequence encoding a polypeptide exhibiting GA 20-oxidase activity.
Examples of a DNA sequence according to the invention are the open reading frames of the sequences shown in SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 or an equivalent sequence through the degeneracy of the genetic code. Thus, a DNA sequence according to the invention may be one which codes for the amino acid sequence shown in SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6. It will be well understood that the invention includes derivatives and mutants of the sequences shown in SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5, provided that such derivatives encode essentially similar peptides having essentially the same function as the peptides encoded by the GA 20-oxidase gene described herein. The said derivatives of the DNA sequence according to the invention may be naturally occurring variants or mutants or, especially, they may be artificially created variants or mutants that may be produced specifically or unspecifically by known mutation methods.
Mutation is to be understood as meaning both the deletion or insertion of one or more bases and the substitution of one or more bases, or a combination of these measures. This is the case especially when the said base substitution is accompanied by a silent mutation which does not result in amino acid substitution and thus does not change the chemical structure of the expression product.
The structural gene according to the invention encoding GA 20-oxidase may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate splice junctions functional in plants, which may be obtained from a synthetic or a natural source. The structural gene according to the invention encoding GA 20-oxidase may further be obtained exclusively from naturally occurring or from synthetic sources. It may be obtained, for example, from a genomic or from a cDNA library or constructed entirely by synthetic means.
Another possibility is the construction of a hybrid DNA sequence comprising cDNA and also genomic DNA and/or synthetic DNA. In that case, the cDNA may originate from the same gene as the genomic DNA, or both the cDNA and the genomic DNA may originate from different gene sources. In any case, however, the genomic DNA and/or the cDNA may each be produced individually from the same gene or from different genes.
If the structural gene contains portions of more than one gene, these genes may originate from one and the same organism, from several organisms belonging to different strains or varieties of the same species or different species of the same genus, or from organisms belonging to more than one genus of the same or a different taxonomic unit.
In any event, the DNA sequence is considered to be within the scope of the invention, if the protein encoded has a GA 20-oxidase activity.
The invention also provides a method of preparing a recombinant DNA encoding GA 20-oxidase. The method may include preparing a cDNA library from a suitable source, and screening this library by means of an antibody against GA 20-oxidase or part of its amino acid sequence, or screening the library by testing for catalytic activity characteristic of the GA 20-oxidase or by any other suitable method known in the art. Standard techniques in recombinant DNA technology can be used as part of the method, such as hybridisation using cDNA probes, polymerase chain reaction using degenerate primers, and restriction fragment length polymorphism.
The method of preparing a recombinant DNA encoding GA 20-oxidase may include preparing a genomic or a cDNA gene library that can be produced by customary routine methods very well known to the person skilled in that field. The basic methods of producing genomic or cDNA gene libraries are described in detail, for example, in Maniatis et al (1982), while information relating to the transfer and application of those methods to plant systems will be found, for example, in the Mohnen (1985) reference [Mohnen et al, EMBO J., 4: 1631-1635 (1985)].
Genomic DNA and cDNA can be obtained in various ways. Genornic DNA, for example, can, using known methods, be extracted from suitable cells and purified.
In a specific embodiment of the present invention, the starting material used for the production of cDNA is generally mRNA, which can be isolated from selected cells or tissues, but especially from cells or tissues of immature seeds of Cucurbitaceae plants such as, for example, C. maxima, which are known to be a rich source of GA biosynthetic enzymes. A further suitable source of GA biosynthetic enzymes is the shoot tissue of Arabidopsis thaliana plants. The isolated mRNA can then be used in a reverse transcription as the matrix for the production of a corresponding cDNA.
The methods of isolating poly(A+) RNA and of producing cDNA are known to the person skilled in the art and are described in detail below in the Examples.
The extracted and purified DNA preparations are then cleaved into fragments for the subsequent cloning. The genomic DNA or cDNA to be cloned may be fragmented to a size suitable for insertion into a cloning vector either by mechanical shearing or, preferably, by cleavage with suitable restriction enzymes. Suitable cloning vectors which are already being used as a matter of routine for the production of genomic and/or cDNA gene libraries include, for example, phage vectors, such as the xcex Charon phages, or bacterial vectors, such as the E. coli plasmid pBR322. Further suitable cloning vectors are known to the person skilled in the art and may be obtained from commercial sources such as, for example, that contained in the xe2x80x98Fast Trackxe2x80x99 mRNA isolation kit obtainable from INVITROGEN or the xcexgt11 Cloning Kit of Amersham.
From the gene libraries produced in that manner, suitable clones comprising the desired gene or parts thereof can then be identified in a screening program, for example with the aid of suitable oligonucleotide probes (probe molecule), and then isolated. Various methods are available for identifying suitable clones, for example differential colony hybridisation or plaque hybridisation. Immunological detection methods based on identification of the specific translation products may also be used.
There may be used as probe molecule, for example, a DNA fragment that has already been isolated beforehand from the same gene or from a structurally related gene and that is capable of hybridisation with the corresponding section of sequence within the desired gene that is to be identified.
Provided that the amino acid sequence of the gene to be isolated or at least parts of that sequence are known, a corresponding DNA sequence can be drawn up on the basis of that sequence information. On the basis of that information it is thus possible to draw up oligonucleotide molecules that can be used as probe molecules for the identification and isolation of suitable clones by hybridising the said probe molecules with genomic DNA or cDNA in one of the methods described above.
In order to facilitate detection of the desired gene, the above-described DNA probe molecule can be labelled with a suitable readily detectable group. Within the scope of this invention, a detectable group is to be understood as being any material having a particular readily identifiable physical or chemical property.
Such materials are already widely used especially in the field of immunoassays, and the majority of them may also be employed in the present Application. Special mention may be made at this point of enzymatically active groups, for example enzymes, enzyme substrates, coenzymes and enzyme inhibitors, and also of fluorescent and luminescent agents, chromophores and radioisotopes, for example, 3H, 35S, 32p, 125I, and 14C. The ready detectability of these labels is based on the one hand on their inherent physical properties (e.g. fluorescent labels, chromophores, radioisotopes) and on the other hand on their reaction and binding properties (e.g. enzymes, substrates, coenzymes, inhibitors).
Also suitable as a probe molecule is a single-stranded cDNA derived from a poly(A)+ RNA, which in turn is isolated from a tissue or a cell known to contain high levels of GA biosynthetic enzymes.
For example, the cDNA sequence of the present invention may be used to isolate genomic or further cDNA sequences encoding GA 20-oxidase. Where a partial cDNA has been obtained, the partial cDNA may be used as a probe to screen the cDNA library in order to isolate a full length cDNA clone. Hybridizing clones are purified, restriction mapped and sequenced. A full length clone will be near message size as well as having a complete open reading frame. To isolate a genomic clone, the full length cDNA is used as a probe to screen a genomic library. By restriction mapping and hybridization to the cDNA, the coding region of the genomic clone is identified. The area upstream from the coding area of the clone is the promoter region.
General methods relating to hybridisation are described, for example, in Maniatis T. et al (1982) and in Haymes B. T. et al (1985) [Haymes B. T. et al, Nucleic Acid Hybridisation: a Practical Approach, IRL Press, Oxford, England (1985)].
Those clones within the above-described gene libraries which are capable of hybridisation with a probe molecule and which can be identified by means of one of the above-mentioned detection methods can then be analysed further in order to determine in detail the extent and nature of the coding sequence.
An alternative method of cloning genes is based on the construction of a gene library composed of expression vectors. In that method, analogously to the methods already described above, genomic DNA, but preferably cDNA, is first isolated from a cell or a tissue capable of expressing a desired gene productxe2x80x94in the present case GA 20-oxidasexe2x80x94and is then spliced into a suitable expression vector. The gene libraries so produced can then be screened using suitable measures, preferably using antibodies, and those clones selected which comprise the desired gene or at least part of that gene as an insert.
Alternatively, total DNA from the DNA library, preferably from the cDNA library, can be prepared and used as a template for a PCR reaction with primers representing low degeneracy portions of the amino acid sequence. Preferably, the primers used will generate PCR products that represent a significant portion of the nucleotide sequence. The PCR products can be further probed to determine if they correspond to a portion of the GA 20-oxidase gene using a synthetic oligonucleotide probe corresponding to an amino acid fragment sequence located in the interior or middle region of the GA 20-oxidase protein.
The cDNA clones and PCR products prepared as described above or fragments thereof may be used as a hybridization probe in a process of identifying further DNA sequences from a homologous or a heterologous source organism encoding a protein product that exhibits GA 20-oxidase activity such as, for example, a fungi or a monocotyledonous plant. A suitable source would be developing tissue from maize or wheat plants.
They may also be used as a RFLP marker to determine, for example, the location of the GA-20 oxidase gene or a closely linked trait in the plant genome or for marker assisted breeding [EP-A 306,139; WO 89/07647].
Using the methods described above it is thus possible to isolate a gene that codes for a GA 20-oxidase.
For further characterisation, the DNA sequences purified and isolated as described above are subjected to sequence analysis. The previously isolated DNA is first cleaved into fragments by means of suitable restriction enzymes and then cloned into suitable cloning vectors, for example the M13 vectors mp 18 and mp 19. The sequencing is carried out in the 5xe2x80x2 to 3xe2x80x2 direction, the dideoxynucleotide chain termination method according to Sanger [Sanger et al, 1977] or the method according to Maxam, and Gilbert [Maxam and Gilbert, 1980] or a commercially available nucleotide sequencing instrumentation [available from Applied Biosystems, Foster City, Calif. and Dupont, Wilmington, Del.] preferably being used. In order to avoid errors in sequencing, it is advantageous to sequence the two DNA strains in parallel. The analysis of the nucleotide sequence and of the corresponding amino acid sequence is advantageously computer-assisted using suitable commercially available computer software [e.g. GCG software of the University of Wisconsin].
The area upstream from the coding area of the clone is the promoter region. The GA 20-oxidase promoter region may be more precisely mapped through deletion analysis. 5xe2x80x2 deletions of a GA 20-oxidase promoter are made by introducing restriction sites by PCR using oligonucleotide primers with restriction sites at the 5xe2x80x2 ends and promoter sequences at the 3xe2x80x2 ends. The PCR products are digested, purified, and cloned into a suitable cloning vector such as, for example, into pBI101 (Clontech). The deletion mutants contain the 5xe2x80x2 untranslated leader sequence fused to the translational start site of the GUS gene. Internal and 3xe2x80x2 deletions of the GA 20-oxidase promoter are made by PCR in a similar manner. The PCR fragments are fused to a GUS Vector containing the CAMV 35S minimal promoter [xe2x88x9246 to +1; Benfey et al, 1990]. Transgenic plants are tested with the GUS fluorometric and histochemical assay.
The GA 20-oxidase promoter region may be suitably used within the scope of the present invention for the preparation of recombinant, or chimaeric, DNA constructs comprising a GA 20-oxidase structural gene, which may be of homologous or of heterologous origin relative to the promoter sequence.
The present invention thus further comprises recombinant DNA sequences comprising, in a 5xe2x80x2 to 3xe2x80x2 direction, a promoter region obtainable from a GA 20-oxidase genomic DNA sequence, which is operatively linked to a GA 20-oxidase coding DNA sequence, which may be homologous or heterologous relative to the promoter sequence. The recombinant DNA sequences result in expression of the associated homologous or heterologous GA 20-oxidase in transformed plant material.
In principle, the DNA can also be prepared by chemical synthesis.
In another aspect, the invention provides a recombinant polypeptide exhibiting GA 20-oxidase activity. This polypeptide or enzyme is soluble and 2-oxoglutarate-dependent. It is capable of acting on, for example, one or more of the following substrates: GA12, GA53, GA15 (open or closed lactone), GA44 (open or closed lactone), GA24, GA19 and GA23. The GA20-oxidase may be derived from plants or fungi, preferably from monocotyledonous and dicotyledonous plants respectively, and more preferably from dicotyledonous plants. A particularly suitable source is plants of the family Cucurbitaceae, such as C. maxima, of which the immature seeds are a convenient source. A further suitable source is plants of the family Cruciferae, such as Arabidopsis thatiana, of which shoot material is a convenient source. In particular, the recombinant GA 20-oxidase is derived from C. maxima or Arabidopsis thaliana respectively, or is a protein having substantial homology thereto (as defined above).
An embodiment of this latter aspect of the invention is a GA 20-oxidase having the amino acid sequence shown in SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6. The invention also includes a protein having substantial homology (as defined above) with this amino acid sequence and having GA 20-oxidase activity. Modified proteins derived from this amino acid sequence by mutation, i.e. addition, substitution or deletion of one or more amino acid residues, and having GA 20-oxidase activity, are also included within the scope of the invention.
Once having identified and isolated the DNA encoding a polypeptide product exhibiting GA 20-oxidase activity, a purified protein can be obtained from transgenic expression of the said DNA, i.e., placing a recombinant DNA comprising a DNA sequence coding for a protein exhibiting GA 20-oxidase activity into an appropriate bacterial, yeast, plant or other suitable cell expression system.
Suitable hosts include bacteria such as E. coli and yeast, including the strain Saccharomyces cerevisiae. Other suitable expression system hosts include insect cells grown in culture. These insect cells may be infected with a baculovirus containing a recombinant DNA molecule according to the invention.
Alternatively, the baculovirus may be used to infect the cells of a living insect, and the insect cells used as the expression system host. The expression system host is then allowed to produce an expression supernatant. This allows facile generation of large amounts of purified recombinant GA 20-oxidase by isolating the enzyme from the expression supernatant
A further object of the present invention is chimaeric gene constructions comprising, in addition to the DNA sequence according to the invention encoding GA 20-oxidase, expression signals which include both promoter and terminator sequences and other regulatory sequences of the 3xe2x80x2 and 5xe2x80x2 untranslated regions and which are operably linked to the coding DNA sequence such as to ensure the expression of the corresponding gene product in the respective host organism.
Suitable control sequences that are preferred within the scope of the invention are those comprising promoter and 5xe2x80x2 and 3xe2x80x2 untranslated regulatory sequences that are functional in plants. These sequences may, independently, be derived from any source, such as, for example, virus, plant or bacterial genes. These promoters or regulatory sequences can be constitutive in nature or can be regulated in their patterns of expression. Such regulation may be temporal or spatial and include developmentally regulated promoters and inducible promoters. Proteins may be optionally expressed in the vacuole or extracellularly using methods well-known in the art (EP 462,065).
In general, any promoter and any terminator capable of bringing about an induction of the expression of a coding DNA sequence (structural gene) may be used as a constituent of the chimaeric gene sequence according to the invention. The said expression signals may promote continuous and stable expression of the gene. Especially suitable are expression signals originating from genes of plants or plant viruses. Examples of suitable promoters and terminators are those of the Cauliflower Mosaic Virus genes (CaMV) or homologous DNA sequences that still have the chacteristics properties of the mentioned expression signals. Also suitable are bacterial expression signals, especially the expression signals of the nopaline synthase genes (nos) or the opine synthase genes (ocs) from the Ti-plasmids of Agrobacteriun tumefaciens. Also to be mentioned here are, for example, ubiquitine promoters, actin promoters, histone promoters and tubulin promoters. Other suitable promoters are an amylase promoter (a-amylase promoter) and an ABA (abscisic acid) inducible promoter.
In a further embodiment of the invention, a promoter region may be used that is obtainable from a GA 20-oxidase genomic DNA sequence as described hereinbefore.
Within the scope of this invention, preference is given to the 35S and 19S expression signals of the CaMV genome or their homologues which can be isolated from the said genome using molecular biological methods, as described for example, in Maniatis et al (1982), and linked to the coding DNA sequence.
Further preferred are expression signals that comprise tissue-preferential or tissue-specific promoters. The term tissue-preferential promoter is used to indicate that a given expression signal will promote a higher level of transcription of an associated expressible DNA, or of expression of the product of the said DNA as indicated by any conventional RNA or protein assay, or that a given DNA sequence will demonstrate some differential effect; i.e., that the transcription of the associated DNA sequences or the expression of a gene product is greater in some tissue than in all other tissues of the plant. For example, the tissue-preferential promoter may direct higher expression of an associated gene product in leaves, stems, roots and/or pollen than in seecl One example of a tissue-preferential promoter, which may be suitably used within the scope of the present invention, is a pith-preferred promoter isolated from a maize TrpA gene.
The term tissue-specific promoter is used to indicate that a given regulatory DNA sequence will promote transcription of an associated expressible DNA sequence entirely in one or more tissues of a plant, or in one type of tissue, while essentially no transcription of that associated coding DNA sequence will occur in all other tissues or types of tissues of the plant. Numerous promoters whose expression are known to vary in a tissue specific manner are known in the art. One such example is the maize phosphoenol pyruvate carboxylase [PEPC], which is green tissue-specific [Hudspeth R. L. and Grula J. W., 1989]. Other green tissue-specific promoters include chlorophyll a/b binding protein promoters and RubisCo small subunit promoters. Further to be mentioned here are, for example, pollen-specific promoters such as those obtainable from a plant calcium-dependent phosphate kinase [CDPK] gene.
A developmentally regulated promoter can also be used. Of course, in the present invention, any promoter which is functional in the desired host plant can be used to direct the expression of an associated gene.
In general, the GA 20-oxidase structural gene may be linked to the promoter region in either a sense or an anti-sense orientation.
It is often advantageous to incorporate a leader sequence between the promoter sequence and the adjacent coding DNA sequence, the length of the leader sequence being so selected that the distance between the promoter and the DNA sequence according to the invention is the optimum distance for expression of the associated structural gene. Suitable leader sequences include leader sequences of various lengths isolated from the 35S CaMV gene (Pierce et al., 1987). The preferred leader sequences are those isolated from the 35S CaMV gene, having a length from about 50 to about 130 nucleotides. The identification of other leader sequences is known in the art. See Della-Cioppa et al, 1987; Schekman, 1985.
Further regulatory DNA sequences that may be used for the construction of chimaeric genes include, for example, sequences that are capable of regulating the transcrption of an associated DNA sequence in plant tissues in the sense of induction or repression.
There are, for example, certain plant genes that are known to be induced by various internal and external factors, such as plant hormones, heat shock, chemicals, pathogens, oxygen deficiency, light, stress, etc.
Another class of genes that are suitable in plants comprises the light-regulated genes, especially the nuclear-coded gene of the small subunit of ribulose-1,5-biphosphate carboxylase (RUBISCO). Morelli et al (1985) have shown that the 5xe2x80x2-flanidng sequence of a RUBISCO gene from the pea is capable of transferring light-inducibility to a reporter gene, provided the latter is linked in chimaeric form to that sequence. It has also been possible to extend this observation to other light-induced genes, for example the chlorophyll-a/b-binding protein.
A further group of regulatable DNA sequences comprises chemically regulatable sequences that are present, for example, in the PR (pathogenesis-related) protein genes of tobacco and are inducible by means of chemical regulators such as those described in EP-A-332,104.
In a specific embodiment of the invention a promoter of the Arabidopsis PR1a gene is being used.
The regulatable DNA sequences mentioned by way of example above may be of both natural and synthetic origin, or they may comprise a mixture of natural and synthetic DNA sequences.
The recombinant DNA sequences of the present invention may further comprise a signal sequence, which is operatively linked to the coding DNA sequence. The signal sequence is responsible for specialized transport of the associated peptide within the plant cell.
The signal sequence of the present invention may be any DNA sequence which is able to direct the transport of an associated polypeptide into one or more of the cellular compartments. The signal sequence is preferably a sequence which is translated into a signal peptide, which becomes separated from the peptide after transit of the peptide is complete. Signal sequences are useful for directing the polypeptide product of the coding DNA sequence to a desired location within the cell, such as to the mitochondria or to the endoplasmic reticulum, or to direct extracellular transport outside of the cell.
To be mentioned here are, for example, N-terminal signal peptides, which are involved in intracellular transport and which can be found at the N-terminal end of proteins transported via the endomembrane system. These signal sequences ensure that the said proteins first pass into the endoplasmic reticulum, where the signal peptide is split off proteolytically from the precursor protein as soon as it has fulfilled its function. By virtue of its specific function, this type of signal peptide sequence has been conserved to a high degree during evolution in all living cells, irrespective of whether they are bacteria, yeasts, fungi, animals or plants.
At the C-terminal end of vacuolar proteins, on the other side, sequences may be found that are involved in directing the expression of the associated coding part of the plant vacuole. Examples of these so-called xe2x80x98vacuolar targetingxe2x80x99 sequences are provided, for example, in EP-A 462,065.
Moreover, the DNA molecule may comprise further sections of sequence that code for peptide fragments which as a whole contribute towards improving the competence for admission into the vacuole, for example the propeptide fragment discovered by Matsuoka K. and Nakamura K. in the N-terminal extension of sporamine [Matsuoka K. and Nakamura K. (1991)].
Further signal sequences useful for the present invention are, for example, the signal sequence from the pathogenesis-related gene (PR-1) of tobacco, which is described in Comellisen et al, 1986; the yeast mitochondrial presequence; Schmitz et al, 1989; the signal sequence from plant mitochondrial Rieske iron-sulfur protein, Huang et al, 1991; mitochondrial and chloroplast targeting peptides, von Heijne et al, 1989.
The present invention therefore also includes chimaeric genetic constructions that comprise, in operable linkage with a structural gene encoding GA 20-oxidase, further regulatory sections of DNA sequence permitting, for example, specifically controlled induction or repression of gene expression.
As a modification of the above aspect, the invention also provides a chimaeric gene construct comprising at least a part of a reverse GA 20-oxidase nucleotide sequence, having at its 5xe2x80x2-end a promoter capable of causing the reverse sequence to express antisense mRNA within a plant and, optionally, further regulatory DNA sequences such as those mentioned above.
The various sections of the chimaeric DNA sequences according to the invention may be linked to one another by methods known per se to form a complete coding DNA sequence. Suitable methods include, for example, the in vivo recombination of DNA sequences having homologous sections and the in vitro linking of restriction fragments.
In the above in vivo and/or in vitro processes for assembling the different sections of the said functional unit, cloning vectors may be involved such as, for example, plasmid or virus (bacteriophage) vectors having replication and control sequences originating from species that are compatible with specific host cells.
The cloning vector generally carries an origin of replication, especially an origin of replication that is capable of functioning in E. coli, in Agrobacterium or in both, and, in addition, specific genes that lead to phenotypic selection features in the transformed host cell, especially to resistance to antibiotics or to specific herbicides. The transformed vectors can be selected on the basis of those phenotypic markers after transformation in a host cell.
The cloning vectors and the host cell transformed with those vectors are generally used to increase the number of copies of the constructs cloned therein. With an increased number of copies it is possible to isolate the vector carrying the chimaeric gene construction and prepare it, for example, for insertion of the chimaeric gene sequence into a plant cell.
Especially suitable within the scope of the present invention are so-called shuttle vectors, which can stably replicate not only in one but in at least two different host organisms such as, for example, in E. coli and in Agrobacterinum tumefaciens, in the presence of a suitable selection marker.
Selectable phenotypic markers that may be used within the scope of this invention include, for example, resistance to ampicillin, tetracycline, hygromycin, kanamycin, methotrexate, G418 and neomycin, but this list, which is given by way of example, is not intended to limit the subject of the invention.
Suitable host cells within the scope of this invention are prokaryotes, including bacterial hosts, for example A. tumefaciens, E. coli, S. typhimuriwn and Serratia marcescens, and also cyanobacteria. Eukaryotic hosts, such as yeasts, mycelium-forming fungi and plant cells, may also be used within the scope of this invention.
The splicing of the chimaeric gene construction according to the invention into a suitable cloning vector is carried out using standard methods, such as those described, for example, in Maniatis et al (1982) and Sambrook et al (1989).
In a further process step, the cloned structural gene coding for GA 20-oxidase may be introduced into one of the commonly used plant transformation cassettes and transformed into a plant cell using standard techniques and, optionally, integrated into the plant genome.
The detection of transformed plant cell may be accomplished using suitable selection systems.
Very convenient selection systems that are preferably applied in transient expression systems are those that are based on a scorable marker such as, for example, regulatory or structural genes controlling anthocyanin biosynthesis, GUS (xcex2-glucuronidase), luciferase, opine synthetases, thaumatin, xcex2-galactosidase, unique synthetic epitopes designed for easy detection by ELISA, phycobiliproteins and various fluorogenic substances.
In a specific embodiment of the present invention use is made of the xe2x80x98GUSxe2x80x99-based marker system, which involves a DNA sequence encoding a xcex2-glucuronidase enzyme operably linked with one or more of the expression signals listed above. Upon expression of the GUS gene in the plant cell the xcex2-glucuronidase enzyme may react with its specific substrate, which leads to the appearance of blue spots that can be easily detected in the plant tissue.
In a further embodiment of the present invention the use is made of coding sequences for the anthocyanin regulatory genes known in the art as Cl and B-Peru [Goff et al, 1990]. Such coding sequences, operably linked to one or more of the several constitutive promoters listed above, can be used to isolate transformants on the basis of the red pigmentation of cells transformed with such genes. The xe2x80x98anthocyaninxe2x80x99-based marker system, on the other hand, involves a red colour reaction.
In a further aspect, the invention provides a transformed host cell comprising recombinant DNA encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with expression signals including promoter and termination sequences which permit expression of said DNA in the host cell. Where the host cell is a plant cell, transgenic plants can be obtained. Thus, the invention provides for the first time a transgenic plant with an altered GA biosynthetic pathway, in particular one which contains and is capable of expressing a recombinant GA:2-oxoglutarate dioxygenase gene. Thus, there is provided a transgenic plant comprising a recombinant DNA encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with plant expression signals including promoter and termination sequences which permit expression of said DNA in the plant.
A modification of the above aspect of the invention is the transformation of a plant with a construct containing a reverse GA 20-oxidase nucleotide sequence (the entire coding sequence or a part thereof) for transcription of antisense mRNA and consequent reduced expression of the GA 20-oxidase gene. Examples of antisense technology are provided in EP-A 240 208 (Calgene) and EP-A 458 367 (Calgene). The reverse nucleotide sequence may be in association with a promoter which is specific to certain plant tissues and/or to external stimulus (e.g. light, cold, heat, chemicals etc.). Another possible means of reducing expression is for example the use of ribozyme technology as described in EP-A 321 201 or WO 89/05852. A combination of antisense and ribozyme technology may also be used within the scope of the present invention for regulating GA 20-oxidase activity.
Also an overexpression of the GA 20-oxidase gene in plants may result in reduced levels of biologically acitve gibberellins in plants.
The invention includes progeny or propagules, including seed, of transgenic plants as defined above. The invention also includes methods of making such transgenic plants.
The recombinant DNA according to the invention comprising the GA 20-oxidase encoding DNA sequence can be introduced into the plant cell in a number of ways that are well known to those of skill in the arts For example, methods of transforming plant cells include microinjection [Crossway et al (1986); Neuhaus (1987)], electroporation [Riggs et al (1986)], Agrobacterium mediated transformation [Hinchee et al (1988)], direct gene transfer [Paszkowsli et al, (1984)], and ballistic particle acceleration using, for example, devices available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. [see, for example, Sanford et al, U.S. Pat. No. 4,945,050; and McCabe et al, (1988). Also see, Weissinger et al (1988); Sanford et al (1987) (onion); Christou et al (1988) (soybean); McCabe et al (1988) (soybean); Datta et al (1990) (rice); Klein et al (1988) (maize); Klein et al (1988) (maize); Klein et al (1989) (maize); Fromm et al (1990); Gordon-Kamm et al (1990) (maize)].
One possible method for introducing genetic material into plant cells comprises, for example, bringing plant cells into contact with viruses or with Agrobacteinum comprising the DNA to be introduced. This may be achieved by infecting sensitive plant cells or by co-cultivating protoplasts derived from plant cells. Within the scope of this invention, Cauliflower Mosaic Virus (CaMV) may be used as a vector for the insertion of the GA 20-oxidase-encoding DNA sequence according to the invention into a plant.
Another method of inserting GA 20-oxidase-encoding DNA sequence into a cell makes use of the infection of the plant cell with Agrobacterium tumefaciens and/or Agrobacteriun rhizogenes, which has previously been transformed with the said gene construction. The transgenic plant cells are then cultured under suitable culture conditions known to the person skilled in the art, so that they form shoots and roots and whole plants are finally formed.
A further possible method of transforming plant material comprises mixed infection using both Agrobacterium rhizogenes and transformed Agrobacterium tumefaciens, as described by Petit et al (1986) for the transformation of carrots.
The GA 20-oxidase-encoding DNA sequence according to the invention can therefore be transferred into suitable plant cells by means of, for example, the Ti-plasmid of Agrobacterium tumefaciens or the Ri-plasmid of Agrobacterium rhizogenes. The Ti-plasmid or Ri-plasmid is transferred to the plant in the course of infection by Agrobacterium and integrated in stable manner into the plant genome.
Any T-DNA-containing vector that can be transferred into plant cells and permits selection of the transformed cells is suitable for use within the scope of this invention such as, for example, a shuttle vector that comprises the GA 20-oxidase-encoding DNA sequence according to the invention cloned in between the left border sequence (LB) and the right border sequence (RB) and that is capable of stable replication both in E. coli and in A. tumefaciens. Preferred is a so-called binary vector system.
Using newly developed transformation techniques, it has also become possible in principle to transform in vitro plant species that are not natural host plants for Agrobacterium. For example, monocotyledonous plants, especially the cereal species and various grasses, are not natural hosts for Agrobacterium.
It has become increasingly evident that monocotyledons can also be transformed using Agrobacterium, so that, using new experimental formulations that are now becoming available, cereals and grass species are also amenable to transformation [Grimsley N.H et al (1987)].
One of the preferred methods for introducing DNA into a plant cell by means of Agrobacterium is the so-called leaf disk transformation using Agrobacterium [Horsch et al (1985)]. Sterile leaf disks from a suitable target plant are incubated with Agrobacterium cells comprising one of the GA 20-oxidase-encoding DNA sequence according to the invention, and are then transferred into or onto a suitable nutrient medium. Especially suitable, and therefore preferred within the scope of this invention, are LS media that have been solidified by the addition of agar and enriched with one or more of the plant growth regulators customarily used, especially those selected from the group of the auxins consisting of a-naphthylacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinic acid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from the group of the cytokinins consisting of kinetin, 6-benzyladenine, 2-isopentenyladenine and zeatin. The preferred concentration of auxins and cytokinins is in the range of from 0.1 mg/l to 10 mg/l.
After incubation for several days but preferably after incubation for 2 to 3 days at a temperature of from 20C to 40C, preferably from 23C to 35C and more especially at 25C and in diffuse light, the leaf disks are transferred to a suitable medium for the purpose of shoot induction. Especially preferred for the selection of the transformants is an LS medium that does not contain auxin but contains cytokinin instead, and to which a selective substance has been added dependent on the marker gene used. The cultures are kept in the light and are transferred to fresh medium at suitable intervals, but preferably at intervals of one week. Developing green shoots are cut out and cultured further in a medium that induces the shoots to form roots. Especially preferred within the scope of this invention is an LS medium that does not contain auxin or cytokinin but to which a selective substance has been added for the selection of the transformants.
In addition to Agrobacterium-mediated transformation, within the scope of this invention it is possible to use direct transformation methods for the insertion of the gene constructions according to the invention into plant material.
Possible methods for the direct transfer of genetic material into a plant cell comprise, for example, the treatment of protoplasts using procedures that modify the plasma membrane, for example, polyethylene glycol treatment, heat shock treatment or electroporation, or a combination of those procedures [Shillito et al (1985)].
In the electroporation technique, plant protoplasts together with plasmids that comprise the GA 20-oxidase-encoding DNA sequence are subjected to electrical pulses of high field strength. This results in a reversible increase in the permeability of biomembranes and thus allows the insertion of the plasmids. Electroporated plant protoplasts renew their cell wall, divide and form callus tissue. Selection of the transformed plant cells can take place with the aid of the above-described phenotypic markers.
A further method for the direct introduction of genetic material into plant cells, which is based on purely chemical procedures and which enables the transformation to be carried out very efficiently and rapidly, is described in Negrutiu I. et al (1987).
Also suitable for the transformation of plant material is direct gene transfer using co-transformation (Schocher R. J. et al 1986).
Co-transformation is a method that is based on the simultaneous taking up and integration of various DNA molecules (non-selectable and selectable genes) into the plant genome and that therefore allows the detection of cells that have been transformed with non-selectable genes.
Further means for inserting genetic material contained in a vector directly into a plant cell comprise using purely physical procedures, for example by microinjection using finely drawn micropipettes [Neuhaus et al (1987)] or by, bombarding the cells with micro projectiles that are coated with the transforming DNA [xe2x80x9cMicroprojectile Bombardmentxe2x80x9d; Wang Y-C et al (1988)] or are accelerated through a DNA containing solution in the direction of the cells to be transformed by a pressure impact thereby being finely atomized into a fog with the solution as a result of the pressure impact [EP-A-434,616].
Microprojectile bombardment has been advanced as an effective transformation technique for cells, including cells of plants. In Sanford et al (1987) it was reported that microprojectile bombardment was effective to deliver nucleic acid into the cytoplasm of plant cells of Allium cepa (onion). Christou et al (1988) reported the stable transformation of soybean callus with a kanamycin resistance gene via microprojectile bombardment. Christou et al reported penetration at approximately 0.1% to 5% of cells. Christou further reported observable levels of NPTII enzyme activity and resistance in the transformed calli of up to 400 mg/l of kanamycin. McCabe et al (1988) report the stable transformation of Glycine max (soybean) using microprojectile bombardment. McCabe et al further report the recovery of a transformed R1 plant from an Ro chimaeric plant.
The transformation of maize plants, including elite maize plants, by microprojectile bombardment can be carried out according to the general protocol described for example in EP-A 478 502, the disclosure of which is incorporated herein by reference.
The list of possible transformation methods given above by way of example is not claimed to be complete and is not intended to limit the subject of the invention in any way.
The present invention therefore also comprises transgenic plant material, selected from the group consisting of protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules, zygotes, etc. and especially, whole and preferably phenotypically normal plants, that has been transformed by means of the processes described above and comprises the recombinant DNA according to the invention in expressible form, and processes for the production of the said transgenic plant material.
Preferred within the present invention are monocotyledonous plants including seed and the progeny or propagueles thereof, but especially graminaceous monocots such as, for example, Lolium, Zea, Triticum, Triticate, Sorghum, Saccharun, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria. Especially preferred are transgenic maize, wheat, and barley plants and seed thereof. Most preferred is the Zea nays Elite inbred line Funk 2717.
Screening of plant cells, tissue and plants for the presence of specific DNA sequences may be performed by Southern analysis (Southern, 1975). Details of this procedure are given in Maniatis et al (1982). This screening may also be performed by the use of Polymerase Chain Reaction procedures (PCR). PCR procedures are described in detail in Mullis et al (1987) and EhrlichA(1989).
Transformation of the plant cells includes separating transformed cells from those that have not been transformed One convenient method for such separation or selection is to incorporate into the material to be inserted into the transformed cell a gene for a selection marker. As a result only those cells that have been successfully transformed will contain the marker gene. The translation product of the marker gene will then confer a phenotypic trait that will make selection possible. Usually the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, e.g., kanamycin, G418, paromomycin, etc., which is placed in a selection media.
Some examples of genes that confer antibiotic resistance include, for example, those coding for neomycin phosphotransferase kanamycin resistance, [Velten et al (1984)]; hygromycin phosphotransferase (hygromycin resistance, [van den Elzen et al (1985)], the kanamycin resistance (NPT II) gene derived from Tn5 Bevan et al (1983); [McBride et al (1990)], the PAT gene described in Thompson et al (1987), and chloramphenicol acetyl-transferase.
An example of a gene useful primarily as a screenable marker in tissue culture for identification of plant cells containing genetically engineered vectors is a gene that encodes an enzyme producing a chromogenic producl One example is the gene coding for production of xcex2-glucuronidase (GUS). This enzyme is widely used and its preparation and use is described in Jefferson (1987).
Once the transformed plant cells have been cultured on the selection media, surviving cells are selected for further study and manipulation. Selection methods and materials are well known to those of skill in the art, allowing one to choose surviving cells with a high degree of predictability that the chosen cells will have been successfully transformed with exogenous DNA.
After transformation of the plant cell or plant using, for example, the Agrobacteriwn Ti-plasmid, those plant cells or plants transformed by the Ti-plasmid so that the enzyme is expressed, can be selected by an appropriate phenotypic marker. These phenotypical markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.
Positive clones are regenerated following procedures well-known in the art. Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed A first evaluation may include, for example, the level of bacterial/fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.
The process for the production of transformed plant material, including whole plants, thus essentially comprises:
first isolating from a suitable source or synthesising by means of known processes a DNA sequence encoding a protein exhibiting GA 20-oxidase activity;
operably linking the said DNA sequence in a 5xe2x80x2 to 3xe2x80x2 direction to plant expression sequences as defined hereinbefore;
transforming the construct of step (b) into plant material by means of known processes and expressing it therein;
screening of the plant material treated according to step (c) for the presence of a DNA sequence encoding a protein exhibiting GA 20-oxidase activity; and optionally regenerating the plant material transformed according to step (c) to a whole and preferably phenotypically normal plant.
The present invention thus also comprises transgenic plants and the sexual andjor asexual progeny thereof, which have been transformed with a recombinant DNA sequence according to the invention.
The expression xe2x80x9casexual or sexual progeny of transgenic plantsxe2x80x9d includes by definition according to the invention all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.
Another object of the invention concerns the proliferation material of transgenic plants. The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
A further aspect of the invention is the provision of an antibody raised against at least a part of the amino acid sequence of GA 20-oxidase. Such antibody is useful in screening a cDNA library in suitable vectors derived from plant tissue RNA.
The GA 20-oxidase gene according to the invention is useful in the modification of growth and developmental processes in transgenic plants. For example, reduced expression with antisense RNA may result in low GA production and therefore decreased elongation growth. The 20-oxidase is a regulatory enzyme and GA production may be particularly sensitive to its activity. It is known to be regulated by day length in long-day rosette plants, such as spinach, in which increased 20-oxidase activity in long days is responsible for bolting. Modifying the expression of this gene may therefore be of particular benefit. Other GA-regulated processes that are potential targets for manipulation are seed germination, flower initiation and development, fruit set and growth and sex expression in some dioecious species.
Thus, in one aspect of the use of this invention, reverse 20-oxidase nucleotide sequences and tissue and/or stimulus (e.g. light, heat, cold, chemical etc.)-specific promoters are used for transformation of plants so as to transcribe antisense mRNA, resulting in reduced expression of the 20-oxidase gene. This method produces plants with reduced endogenous GA levels and consequently altered growth habit and/or other developmental processes.
This method can be used to reduce vegetative growth as in:
straw strengthening in small grain cereals and rice;
for the prevention of lodging;
preventing lodging in oilseed rape and improving its canopy structure;
improving seedling quality for transplantation;
reducing growth of turf and amenity grasses;
reducing shoot growth in orchard and amenity trees; producing ornamental plants with more compact growth habits;
improving tolerance to cold, drought and fungal infection; and
increasing yields by diversion of assimilates from vegetative to reproductive organs.
The method is also useful to prevent bolting and flowering in rosette plants, e.g. sugar beet, lettuce, spinach and brassicas. It is useful to prevent sprouting, as in potato tubers. It is also useful to prevent precocious seed germination.
The invention is also useful in the transformation of plants with constructs containing the 20-oxidase sequence and tissue and/or stimulus-specific promoters for increased expression of the GA 20-oxidase gene. This method will increase the levels of biologically active GAs and so modify plant development, in cases where 20-oxidation is a rate-limiting step. The method can be used to improve fruit-set and growth as in: increasing berry size in seedless grapes (also to increase rachis length and produce a less compact cluster); increasing fruit set in citrus, particularly in clementines; delaying ripening in citrus; improving fruit set in pear and to decrease seed number, and to modify shape of apple fruit and improve skin texture.
The method can potentially be used to increase stem extension and leaf expansion, for example to increase stem length and sugar yield in sugar cane; to increase yield and earliness in celery and rhubarb; to increase yield in cabbage, lettuce, spinach etc.; and to increase forage yields in grasslands. The method can be used to stimulate seed germination, for example in the advancement of malting and increase in malt yields in cereals (e.g. barley, wheat, oats). The method can be used to produce uniform bolting and to stimulate flowering, for example in seed production in lettuce and other rosette species, or in advanced cropping of artichokes. The method can be used to induce flower formation in conifers. It can also be used to overcome dormancy of tubers and to hasten shoot emergence as in potatoes, sweet yams etc. Furthermore, the method can be used to induce staminate flowers in gynoecious species, such as cucumber.
Reference is now made to the accompanying sequence listing and the drawings, in which:
SEQ ID NO 1 shows the nucleotide sequence of GA 20-oxidase cDNA clone pB11 obtained from Curcubita maxima seed.
SEQ ID NO 2 shows the amino acid sequence of the GA 20-oxidase protein corresponding to cDNA clone pB11.
SEQ ID NO 3 shows the nucleotide sequence of GA 20-oxidase cDNA clone pAt2301 obtained from Arabidopsis thaliana. 
SEQ ID NO 4 shows the amino acid sequence of the GA 20-oxidase protein corresponding to cDNA clone pAT2301.
SEQ ID NO 5 shows the nucleotide sequence of GA 20-oxidase cDNA clone pAt2353 obtained from Arabidopsis thaliana. 
SEQ ID NO 6 shows the amino acid sequence of the GA 20-oxidase protein corresponding to cDNA clone pAt2353
SEQ ID NO 7 shows the amino acid sequence of a synthetic peptide that had been produced on the basis of the amino acid sequence of a peptide resulting from trypsin digestion of purified GA12 20-oxidase from C. maxima endosperm.
SEQ ID NOs 8 and 9 show the amino acid sequence of two peptides corresponding to oligodeoxynucleodde primers that are designed based on amino acid regions conserved between the Cucurbita maxima cotyledon gibberellin 20-oxidase and other plant dioxygenases, including the tomato E8 ripening-related protein, tomato ethylene-froming enzyme, hyoscamine 6-hydroxylase from Hyoscyamus niger, barley flavanone 3-hydroxylase and the A2 gene from maize.
SEQ ID NOs 10 and 11 show the sequence of two oligodeoxynucleotide primers that are designed based on amino acid regions conserved between the Cucurbita mama cotyledon gibberellin 20-oxidase and other plant dioxygenases, including the tomato E8 ripening-related protein, tomato ethylene-froming enzyme, hyoscamine 6-hydroxylase from Hyoscyamus niger, barley flavanone 3-hydroxylase and the A2 gene from maize. The upstream and downstream primers contained restriction endonuclease cleavage sites for HindIII and EcoRI, respectively, at their 5xe2x80x2 termini.
SEQ ID NOs 12 and 13 show the nucleotide and the corresponding amino acid sequence of an insert of cDNA clone pAt2204, whose predicted amino acid sequence is 67% identical to that of pumpkin gibberellin 20-oxidase.
SEQ ID NOs 14 to 17 show the nucleotide sequences of four oligonucleotides, which are used in conjunction with the M13 universal sequencing primer in PCR reactions.